Project Summary. Viral respiratory infections caused by rhinovirus (RV) and respiratory syncytial virus (RSV) are the top cause of hospitalization in infants and the most common risk factor of recurrent wheezing and childhood asthma. Understanding the cellular and molecular mechanisms by which some infants develop recurrent wheezing after RV or RSV infection could lead to novel preventive and treatment strategies. To fill this gap, we propose a translational study in human infants that capitalizes on the increasing evidence that the airway epithelium shapes virus-induced airway immune responses and plays a fundamental role in the pathogenesis of asthma. Using a new model of the human infant airway epithelium, we provide novel evidence that airway epithelial cells (AECs) from human infants produce high levels of thymic stromal lymphopoietin (TSLP), an epithelial cytokine that mediates virus-induced type 2 (T2) inflammation and airway hyperreactivity in animal models and in asthmatic individuals. We also observe that: (1) human infant AECs display increased secretion of IL1?, a potent inducer of TSLP via NF-kB signaling; and (2) IL1? elicits stronger TSLP production in infant AECs relative to adult AECs. In agreement with these cellular findings, our in vivo studies demonstrate that: (1) natural RV or RSV infections in human infants are associated with elevated airway TSLP levels that correlate with airway T2 inflammation and IL1? production; and (2) higher virus-induced airway TSLP levels are linked to severe respiratory disease requiring intensive care unit admission and recurrent wheezing within 12 months post-viral infection. Our published and preliminary data strongly support our hypothesis that during infancy the human airway epithelium has enhanced TSLP responses to RV and RSV infections, which increases the risk for severe acute respiratory disease and recurrent wheezing post-viral infection in susceptible infants. Three Specific Aims are proposed: Aim 1. To test the hypothesis that during infancy the human airway epithelium has enhanced TSLP responses to RV or RSV infections and to define the role of IL1? and NF-kB signaling in mediating developmental differences in TSLP production in human infant vs. adult AECs. Aim 2. To test the hypothesis that increased TSLP production (in vivo and ex vivo) in nasal AECs from infants hospitalized with RV or RSV is linked to increased airway T2 inflammation, reduced viral clearance and worse acute respiratory disease. Aim 3. To test the hypothesis that TSLP responses of infant nasal AECs obtained during the first RV or RSV hospitalization are associated with the development of recurrent wheezing in a two- year prospective study. IMPACT. This proposal will move forward our understanding of the infant AECs pathways involved in the pathogenesis of virus-induced wheezing in early life, and will define the role of TSLP in this process. This new knowledge may ultimately lead to interventions to reduce the incidence of recurrent wheezing and asthma in millions of infants undergoing RV and RSV infections.